CN115216447B

CN115216447B - On-line light transparent reagent and transparent method for organ chip

Info

Publication number: CN115216447B
Application number: CN202210822876.9A
Authority: CN
Inventors: 朱; 俞婷婷; 钟响; 李东宇; 朱京谭
Original assignee: Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology
Priority date: 2022-07-06
Filing date: 2022-07-06
Publication date: 2024-05-10
Anticipated expiration: 2042-07-06
Also published as: CN115216447A

Abstract

The invention belongs to the technical fields of organ chips, biomedical engineering and tissue optics, and particularly relates to an on-line light transparent reagent of an organ chip and a transparent method. According to the on-line light transparent reagent suitable for the organ chip, iodixanol solution and proper concentration of metal ions for regulating osmotic pressure in the organ chip and/or maintaining physiological functions of the organ chip are introduced into the reagent, so that the light transparent reagent can not influence functions of the organ chip while the organ chip is transparent, is beneficial to biomedical imaging observation, and can acquire fine structure information of the organ chip with high precision and large flux.

Description

On-line light transparent reagent and transparent method for organ chip

Technical Field

The invention belongs to the technical fields of organ chips, biomedical engineering and tissue optics, and particularly relates to an on-line light transparent reagent of an organ chip and a transparent method.

Background

The organ chip is an emerging scientific technology for realizing the function of simulating organism organs by culturing cells in the in-vitro chip. Through ingenious bionic design, the organ chip can simulate the shape and function of a real organ to a certain extent, can provide a model similar to a real organism for medical, pharmaceutical and life science research, and provides data for substituting animal and clinical experiments for research such as drug research and development, screening, disease model construction and the like. A variety of organ-chips have been developed and applied to specific clinical studies.

Research and development of organ chips are not separated from accurate three-dimensional measurement of artificial micro-organs in the chip. Traditional medical imaging techniques such as CT, MRI, etc. can achieve three-dimensional imaging measurements of real organs, but these methods have too low resolution to be applied to imaging measurements on miniaturized chips. Optical microscopy imaging provides an important means for obtaining structural information of tissue and organs with high resolution. To obtain three-dimensional structure information of biological tissues, the traditional method mostly adopts a tissue slicing mode, firstly cuts a sample into slices of several micrometers to tens of micrometers, and then obtains images with resolution of cells or even subcellular through a fluorescence microscope. And there are a large number of structures of non-biological tissues in the partial organ chip, this slicing method is obviously not very suitable. Another method is to acquire the fine structure information of the target by using an optical slice microscope, such as a confocal laser scanning microscope, a two-photon microscope, a light-sheet illumination fluorescence microscope, or other tomographic imaging techniques, such as OCT.

However, the organ-chip has a multi-layer complex structure, wherein the cultured artificial micro-organ often has a certain thickness, and due to the mismatch of refractive indexes among components, the propagation path of light in a sample is deviated towards all directions, and strong scattering tendency is shown, so that the imaging depth of the optical imaging system is limited, and further the imaging quality of tissues is gradually reduced along with the increase of the depth, and the difficulty of imaging the organ-chip with high quality is increased.

In recent years, the rising tissue light transparency technology balances the refractive indexes of each component of a sample through various physical and chemical strategies, aims to reduce the light scattering phenomenon in the sample, improves the transparency of the sample and enables the sample to become transparent to light, but the existing method is mostly used for isolated real tissues or organs or samples such as zebra fish embryos and the like which are convenient to observe.

For organ-chips, they are extremely sensitive to culture environment, and therefore, there is no suitable on-line transparent method, and long-term in-vivo tracking and observation cannot be performed. In general, when the existing method is used for carrying out high-resolution high-flux observation on an organ chip, a sample is fixed by using a fixing liquid, and various organic or inorganic reagents are adopted for treatment, so that the method cannot be applied to living body online observation.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an on-line optical transparent reagent and a transparent method for an organ chip, which are used for adjusting the osmotic pressure of the organ chip and maintaining the physiological function of the organ chip by introducing metal ions with proper concentration ranges into the optical transparent reagent, so that the technical problems that the long-term living body on-line transparency, tracking observation and the like of the organ chip cannot be carried out in the prior art are solved.

In order to achieve the above purpose, the invention provides an on-line optical transparent reagent of an organ chip, which comprises a culture solution for culturing the organ chip and iodixanol, wherein the mass percentage of the iodixanol in the transparent reagent is 10% -35%;

The transparent reagent also contains metal ions for regulating osmotic pressure of the organ chip and/or maintaining physiological functions of the organ chip; the total concentration of the metal ions ranges from 50 to 150 mM.

Preferably, the metal ion is one or more of potassium ion, calcium ion, magnesium ion, sodium ion and zinc ion.

Preferably, the concentration of potassium ions in the transparent reagent ranges from 1 to 4mM; the concentration of calcium ions ranges from 0.3 to 0.9mM; the concentration of magnesium ions ranges from 0.3 to 0.5mM, the concentration of zinc ions ranges from 0 to 6 mu M, and the concentration of sodium ions ranges from 50 to 120mM.

Preferably, the culture medium is DMEM medium, F12 medium, MEM medium, RPMI1640 medium, or a derivative medium of the above medium.

Preferably, the organ chip is a heart organ chip, and the mass percentage of iodixanol in the transparent reagent is 24% -26.1%; the concentration of calcium ions in the transparent reagent is 0.75-0.85mM, and the concentration of potassium ions is 3.45-3.55mM; the concentration of magnesium ions is 0.35-0.45mM, and the concentration of sodium ions is 55-100mM.

Preferably, the organ chip is a brain-like organ chip, and the mass percentage of iodixanol in the transparent reagent is 17.1% -24%; the transparent reagent has calcium ion concentration of 0.35-0.45mM, potassium ion concentration of 1.95-2.05mM, magnesium ion concentration of 0.35-0.45mM, zinc ion concentration of 4.5-5.5 μm, and sodium ion concentration of 50-100mM.

Preferably, the organ chip is a tumor chip, the mass percentage of iodixanol in the transparent reagent is 27.3% -33.3%, the concentration of calcium ions in the transparent reagent is 0.35-0.45mM, the concentration of potassium ions in the transparent reagent is 1.95-2.05mM, the concentration of magnesium ions is 0.35-0.45mM, and the concentration of sodium ions in the transparent reagent is 70-80mM.

According to another aspect of the present invention, there is provided a method for on-line optical transparency of an organ-chip using the optical transparency reagent, comprising the steps of:

(1) After bubble removal treatment is carried out on the organ chip, pouring the light transparent reagent into the organ chip at the speed of 2-200 mu L/min, and continuously pouring for 0.4-0.6 hour; or immersing the artificial micro-tissue in the organ chip in the light transparent reagent for 0.8-1.2 hours; making the organ chip transparent to obtain a transparent organ chip;

(2) And (3) carrying out on-line observation on the organ chip obtained in the step (1) after the transparent treatment under a microscope.

Preferably, the method further comprises the steps of:

(3) After the observation is finished, the culture solution is poured into the organ chip at the speed of 2-200 mu L/min, and the pouring is continued for 0.4-0.6 hour; or re-soaking the artificial micro-tissue in the organ chip in the culture solution to obtain the recovered organ chip.

In general, the above technical solutions conceived by the present invention have the following beneficial effects compared with the prior art:

(1) The invention provides an on-line light transparent reagent suitable for organ-chip, which is prepared by introducing iodixanol and proper concentration of metal ions for regulating osmotic pressure of the organ-chip and/or maintaining physiological functions of the organ-chip into the reagent, wherein the iodixanol is used for matching the refractive index of cytoplasm and culture environment; the appropriate metal ion type and concentration range ensure that the light transparent reagent can not influence the functions of the organ chip while the organ chip is transparent, thereby being beneficial to biomedical imaging observation and obtaining the fine structure information of the organ chip with high precision and large flux.

(2) The living body on-line optical transparency method for carrying out the organ chip by using the optical transparency reagent is simple, the used materials are simple and easy to obtain, the time required by the preparation process is very short, and the on-line optical transparency method is favorable for the instant observation of the organ chip. The method is simple to operate, low in cost, compatible with various fluorescent markers and imaging methods, and convenient for accurate measurement of organ chips.

(3) The on-line light transparent reagent and the corresponding transparent method of the organ chip make up for the defect of the on-line living body transparent method of the organ chip in the prior art. The transparent method can be effectively combined with various imaging technologies such as confocal imaging, two-photon imaging or light sheet fluorescence microscopic imaging, and provides a solution with high resolution and high flux for acquiring three-dimensional structure information of the artificial micro-organ.

Drawings

FIG. 1 is a schematic diagram showing the composition of an on-line optically transparent reagent of an organ-chip according to the invention.

Fig. 2 is a schematic diagram of a layered structure of an organ-chip according to some embodiments of the invention.

FIG. 3 is a schematic diagram of a first patterning layer, i.e., a culture layer, of a chip pattern according to some embodiments of the invention.

Fig. 4 is a diagram of a second pattern layer, i.e., viewing layer, in accordance with some embodiments of the present invention.

Fig. 5 is a schematic diagram showing the overall structure of an organ-chip according to some embodiments of the invention.

FIG. 6 is a graph showing fluorescence contrast of tumor micro-organs before and after on-line transparency of the tumor organ chip according to example 1 of the present invention.

FIG. 7 is a graph showing the comparison of the tumor micro-organs on the adapted chip before and after on-line transparency of the organ chip according to example 2 of the present invention.

FIG. 8 is a graph showing fluorescence contrast of heart micro-organs before and after on-line transparency of heart organ chips according to example 3 of the invention.

The same reference numbers are used throughout the drawings to reference like elements or structures, wherein:

1-a transparent substrate; 2-a first pattern layer; 21-microfluidic main channel; 22-a first through hole; 23-an intermediate channel; 3-a second pattern layer; 31-a second through hole; 4-a transparent cover sheet layer; 5-a main channel liquid inlet hole; 6-a main channel liquid outlet hole; 7-an intermediate channel liquid inlet hole; 8-an intermediate channel liquid outlet hole; 9-a connection layer; 10-boss.

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

The invention provides an on-line light transparent reagent suitable for organ chips, which is shown in figure 1, and comprises a culture solution for culturing the organ chips and iodixanol, wherein the mass percent of the iodixanol in the transparent reagent is 12% -35%; the transparent reagent also contains metal ions for regulating osmotic pressure of the organ chip and/or maintaining physiological functions of the organ chip; the total concentration of the metal ions ranges from 50 to 150 mM.

When the transparent reagent is prepared, the culture solution and iodixanol solution can be mixed, then metal ions with proper types and concentrations are added, and the iodixanol solution is obtained by dissolving iodixanol in deionized water, and in a preferred embodiment, medical iodixanol injection can be directly adopted. When the transparent reagent is prepared, the transparent reagent can be prepared according to the volume ratio of 60 weight percent of iodixanol solution to the culture solution of 1:4-1:1, and the molecular structure of the iodixanol is shown as a formula (I):

First, the first is

CN110579442a discloses a refractive index matching method for adapting to microfluidic-optical sheet imaging and application thereof, which adopts iodixanol original solution or iodixanol crystal after crystallization, adjusts imaging environment of living body mode biological sample optical sheet imaging, and enables refractive index matching between adjacent media in the imaging environment. The applicant of the present invention initially adopts the method to directly perform on-line optical transparent observation or imaging on an organ chip such as a heart organ chip, a brain organ-like chip or a tumor chip, and finds that cells in a part of the organ chip quickly die, so that on-line transparent observation experiments fail. Through extensive experimental investigation, the applicant of the present invention found that for an organ-chip, the transparent reagent needs to contain metal ions capable of adjusting the osmotic pressure of the organ-chip and/or maintaining the physiological functions of the organ-chip, and ensure that the metal ions are within a proper concentration range, so that on-line transparent observation or imaging of the living body of the organ-chip can be realized. And experiments show that, unlike osmotic pressure and functional requirements of organs in organisms, the organ chip simulates biological environment, but the concentration requirement on metal ions is more severe possibly due to the lack of corresponding homeostasis regulating mechanism in vitro.

In some embodiments, the metal ion is one or more of potassium ion, calcium ion, magnesium ion, sodium ion, and zinc ion.

In a preferred embodiment, the concentration of potassium ions in the transparent reagent is in the range of 1-4mM; the concentration of calcium ions ranges from 0.3 to 0.9mM; the concentration of magnesium ions ranges from 0.3 to 0.5mM, the concentration of sodium ions ranges from 50 to 120mM, and the concentration of zinc ions ranges from 0 to 6 mu M.

The culture solution is used as a basic component of the light transparent reagent, and corresponding cell culture solutions can be selected according to different organ chip types in practical application, and can be classical cell culture media such as DMEM (DMEM medium), F12 (F12) culture medium, MEM (MEM) culture medium, RPMI1640 culture medium, derivative culture media of the classical culture media or other culture solutions. For example, for heart organ chips, RPMI1640 medium is typically selected; for brain-like chips, F12 medium is generally selected; for tumor chips, culture medium is generally selected according to tumor types, and most of culture medium is DMEM and RPMI 1640. The culture solution can be prepared by directly purchasing a liquid culture solution or purchasing a solid culture medium and double distilled water to obtain the culture solution with proper concentration.

When the organ chip is a heart organ chip, the medical iodixanol injection and the culture solution are prepared according to the volume ratio of 1:1.3-1:1.5, preferably 1:1.4; correspondingly, the mass percentage of iodixanol in the transparent reagent is 24% -26.1%, preferably 25%; the concentration of calcium ions in the transparent reagent is 0.75-0.85mM, and the concentration of potassium ions is 3.45-3.55mM; the concentration of magnesium ions is 0.35-0.45mM, and the concentration of sodium ions is 55-100mM. These metal ions and their concentration ranges ensure that the heart micro-organs are free of dysfunction and can discharge and pace normally.

When the organ chip is a brain-like organ chip, the volume ratio of the medical iodixanol injection to the culture solution is 1:1.5-2.5; preferably 1:2, the clear reagent is formulated; then, metal ions are additionally added, so that the concentration of calcium ions in the transparent reagent is 0.35-0.45mM, the concentration of potassium ions is 1.95-2.05mM, the concentration of magnesium ions is 0.35-0.45mM mM, the concentration of sodium ions is 50-100mM, and the concentration of zinc ions is 4.5-5.5 mu M. Experiments show that the lack of potassium, calcium and magnesium can cause dysfunction of the brain-like function, the nerve electric activity can not be normally performed, and the lack of zinc can influence the physiological activity of the brain-like function. Correspondingly, the mass percent of iodixanol in the transparent reagent is 17.1% -24%, preferably 20%.

When the organ chip is a tumor chip, the medical iodixanol injection and the culture solution are prepared according to the volume ratio of 1:0.8-1:1.2, and then metal ions are added, so that the concentration of calcium ions in the transparent reagent is 0.35-0.45mM, the concentration of potassium ions is 1.95-2.05mM, the concentration of magnesium ions is 0.35-0.45mM, the concentration of sodium ions is 70-80mM, and correspondingly the mass percentage of iodixanol in the transparent reagent is 27.3-33.3%.

In some embodiments, experiments show that in addition to the culture solution and iodixanol solution, metal ions are required to be added to the heart organ chip or the brain organ-like chip so as to meet the osmotic pressure and physiological function requirements of the organ chip; the tumor chip has no special functional requirement, and only some metal ions such as sodium ions for maintaining the osmotic pressure of the tumor chip can be added; if the metal ions in the culture solution are sufficient to maintain the osmotic pressure of the tumor chip, no additional addition is needed.

When it is desired to add metal ions additionally to the transparent reagent, the metal ions may be added in the form of metal chlorides. The metal ions to be added can be prepared into a mixed adjusting reagent with higher concentration, and then the adjusting reagent with proper volume is added into the mixed solution of iodixanol and culture solution according to the requirement, and the influence on the concentration of iodixanol in the mixed solution is basically negligible because the prepared reagent has very high concentration and the prepared reagent added into the mixed solution is generally very small in volume.

The invention also provides a method for carrying out on-line optical transparency of the organ chip by using the optical transparency reagent, which comprises the following steps:

(1) After bubble removal treatment is carried out on the organ chip, the light transparent reagent as described above is poured into the organ chip at the speed of 2-200 mu L/min, and the pouring is continued for 0.4-0.6 hours; or immersing the artificial micro-tissue in the organ chip in the light transparent reagent for 0.8-1.2 hours; making the organ chip transparent to obtain a transparent organ chip;

In some embodiments, the method further comprises the step of recovering the organ-chip:

In some embodiments of the present invention, a method for long-term on-line in vivo transparent viewing of an organ-chip is provided, the method comprising the steps of:

S1, mixing 60% of medical iodixanol injection (Sigma) with a culture solution according to a certain proportion, uniformly mixing by a pipetting gun, and adding one or more of calcium chloride, potassium chloride, zinc chloride, sodium chloride and the like to adjust the ion concentration to obtain a transparent reagent.

S2, transparency: and filling the obtained light transparent reagent into a pump, discharging bubbles in the chip, and continuously pouring the light transparent reagent into the chip at the speed of 2-200 mu L/min for half an hour. If the chip used is not or is inconvenient to perfuse, the artificial micro organ (or artificial micro tissue) may be immersed in the optically transparent reagent for one hour. The proper temperature and gas environment should be maintained throughout the process.

S3, observing: after the pouring is finished, the chip can be moved to a microscope for observation.

S4, recovering: and (3) continuously pouring the culture solution into the chip at the speed of 2-200 mu L/min for 60 minutes, and then putting the chip back into the incubator. If the used chip cannot or is inconvenient to perfuse, the artificial micro-organ is restored to the culture solution environment.

For the organ chip with the fluid channel, the optical transparent reagent of the invention can be poured into the fluid channel of the organ chip, so that the artificial micro-tissue in the organ chip is soaked in the optical transparent reagent for transparency or restoration; for the organ chip without the fluid channel, the artificial micro-tissue in the organ chip can be directly soaked in the light transparent reagent for transparency or restoration.

The in-vivo on-line optical transparent reagent and the transparent method for the organ chip provided by the invention can be applied to various organ chips, the organ chip broadly comprises organ chips containing fluid channels and also comprises various artificial micro-organs (also called artificial micro-tissues), and the micro-organs or the organ chips can be purchased from the market or can be cultured and manufactured by self according to the method in the prior art. For culturing three-dimensional artificial micro-organs such as myocardial bulb, tumor bulb, brain-like artificial micro-organs, only cell suspension (50-100 ten thousand per milliliter) is introduced into a chip, and then centrifuged, soaked in culture solution for culturing; for heart organ chip or brain organ-like chip, corresponding cell factor needs to be added during culture, and the culture method of three-dimensional artificial micro-organ in the prior art can be referred to culture the three-dimensional artificial micro-organ in the culture hole of the organ chip.

The living body online light transparent reagent and the transparent method suitable for the organ chip and the transparent tissue sample obtained by the organ chip structure can be used for confocal imaging, two-photon imaging or light sheet fluorescence microscopic imaging to obtain three-dimensional high-resolution structure information of the artificial micro-organ.

In some embodiments of the invention, an organ chip is self-made and used for on-line transparency and on-line observation, through the structural design of the organ chip, the organ chip can be directly observed on line by confocal imaging, two-photon imaging or light sheet fluorescence microscopy imaging after transparency, after the observation is finished, the organ chip is recovered by adopting a culture solution, and after the recovery is finished, the organ chip can be put into use again, so that the function of the organ chip can be maintained all the time.

As shown in fig. 2, an organ chip suitable for on-line transparency and on-line observation according to some embodiments of the present invention is provided with a transparent substrate layer 1, a connection layer 9, a first pattern layer 2, a second pattern layer 3 and a transparent cover layer 4 stacked from bottom to top; the first pattern layer 2 and the second pattern layer 3 respectively adopt PDMS base materials, wherein: the first pattern layer 2 contains a chip pattern of the organ chip. The chip pattern of the first pattern layer 2 is shown in fig. 3, and the chip pattern includes a microfluidic main channel 21 and a plurality of first through holes 22 which are arranged on the microfluidic main channel 21 in a scattered manner and are communicated with the microfluidic main channel 21.

The patterns contained in the second pattern layer 3 comprise a plurality of second through holes 31, and the second through holes 31 in the second pattern layer 3 are in one-to-one correspondence and communicated with the first through holes 22 in the first pattern layer 2; the pattern of the second pattern layer 3 is shown in fig. 4. The overall structure of the organ-a-chip is schematically shown in FIG. 5. During operation, the second through holes 31 in the second pattern layer 3 are used as observation holes, the first through holes 22 in the first pattern layer 2 are used as culture holes of artificial micro-organs, the positions of the first through holes 22 and the second through holes 31 are in one-to-one correspondence and are communicated, separation of the culture holes and the observation holes is realized through mutual matching, and the method is beneficial to reducing the complexity of media between a lens and a sample so as to improve the observation quality.

The bottom of the second pattern layer 3 and the top of the transparent substrate layer 1 are respectively connected with the top and the bottom of the microfluidic main channel 21, so that the microfluidic main channel 21 becomes a closed channel.

The two ends of the second pattern layer 3 are protruding with respect to the transparent cover plate layer 4, and the protruding portions at the two ends are respectively provided with a main channel liquid inlet hole 5 and a main channel liquid outlet hole 6, which are communicated with the microfluidic main channel 21, as shown in fig. 3.

The first pattern layer 2 and the second pattern layer 3 are PDMS blocks with different patterns prepared by a soft lithography method. The first pattern layer 2 is manufactured as follows: firstly, drawing a photoetching drawing of a first pattern layer, manufacturing a corresponding mask, forming a corresponding male die on a silicon wafer through soft photoetching, pouring PDMS prepolymer into the silicon wafer, curing for 60min in an oven at 80 ℃, taking down a PDMS block with a formed pattern after curing, and cutting into a proper size to obtain the first pattern layer. The soft lithography is specifically as follows: firstly, photoresist with preset thickness is spin-coated on a silicon wafer, then pre-baking is carried out, a mask is covered on the silicon wafer after the pre-baking is finished, alignment exposure is carried out in a photoetching machine, and post-baking is carried out after the exposure is finished. And then developing and fixing.

The manufacturing method of the second pattern layer comprises the following steps: firstly, drawing a photoetching drawing of a second pattern layer, manufacturing a corresponding mask, forming a corresponding male die on a silicon wafer through soft photoetching, pouring PDMS prepolymer into the silicon wafer, curing for 60min in an oven at 80 ℃, taking down the PDMS block with the formed pattern after curing, and cutting into a proper size to obtain the second pattern layer.

An intermediate channel 23 is further arranged between every two first through holes 22 on the first pattern layer 2; the protruding parts at two ends of the second pattern layer are also provided with a middle channel liquid inlet hole 7 and a middle channel liquid outlet hole 8. The intermediate channels are additionally arranged so that each first through hole 22 not only has fluid passing through from the microfluidic main channel, but also has fluid passing through the intermediate channel in the other direction, and transparent reagent is circularly introduced into the intermediate channel 23, so that the transparent reagent in the intermediate channel slowly enters the culture chamber in the chip by using the diffusion effect, and the influence of the fluid shearing force on the artificial micro-organ in the culture chamber can be reduced. For heart micro-organs, the circulation of transparent reagent from the middle channel can last for 0.5-1h; for tumor micro-organs, the continuous feeding can be carried out for 0.5 to 2 hours; for the brain-like organ, the continuous ventilation is carried out for 6-12h. In a preferred embodiment, the microfluidic main channel and the intermediate channel are perpendicular to each other.

The main channel liquid inlet 5, the main channel liquid outlet 6, the middle liquid inlet 7 and the middle liquid outlet 8 are all boss 10, and the main channel liquid inlet 5, the main channel liquid outlet 6, the middle liquid inlet 7 and the middle liquid outlet 8 are led out by the boss 10, so that liquid inlet and liquid outlet are facilitated. The figure is provided with 6 bosses which respectively correspond to a main channel liquid inlet hole 5, a main channel liquid outlet hole 6, two middle liquid inlet holes 7 and two middle liquid outlet holes 8.

The cross-sectional shapes of the first through hole 22 and the second through hole 31 are the same, and are circular or polygonal, such as hexagonal or octagonal; the bottom of the first through hole 22 is a U-shaped bottom, and the U-shaped bottom is beneficial to three-dimensional culture of cells. The U-shaped bottom can be prepared by the following method: after the first pattern layer 2 is prepared, 0.005-0.015g of uncured PDMS prepolymer is taken by a liquid transfer device, dripped into the first through hole 22 of the first pattern layer 2, and after standing for 3-8 minutes, the PDMS prepolymer can spontaneously form a U-shaped bottom surface under the action of surface tension, and then heated and cured to obtain the first pattern layer with the U-shaped bottom through hole.

The material of the connecting layer 9 is PDMS, and the hardness of the PDMS material adopted by the connecting layer 9 is smaller than that of the PDMS material adopted by the first pattern layer 2, which is favorable for making the first pattern layer 2 firmly connected with the transparent substrate layer 1, and compared with a PDMS-glass interface, the PDMS-PDMS interface is more favorable for spreading PDMS, which is favorable for forming the U-shaped bottom. And at this time, the bottom of the second pattern layer and the top of the connection layer are respectively connected with the top and the bottom of the microfluidic main channel, so that the microfluidic main channel becomes a closed channel. The thickness of the connection layer 9 is in the range of 5-20 μm.

The thickness of the first pattern layer 2 is 1.5 + -0.1 mm, and the thickness of the second pattern layer 3 is 100 + -10 μm. The transparent basal layer 1 is a glass slide or a cover glass; the transparent cover plate layer 4 is a glass slide or a cover glass.

In some preferred embodiments, the cross-sectional shapes of the first through hole 22 and the second through hole 31 are circular, and according to the order of the glass slide-connecting layer-first pattern layer 2-second pattern layer 3-cover glass-material inlet and outlet boss, the parts of each layer are sequentially bonded by using Plasma treatment, the circular through hole positions of the first pattern layer 2 and the second pattern layer 3 are ensured to be in one-to-one correspondence, and finally corresponding microfluidic facilities are connected, so that the overall structure of the obtained microfluidic organ chip is schematically shown in fig. 5. The organ chip design adapting to the transparent reagent and the transparent method solves the problems of long time, large deformation and the defect of the on-line organ chip transparent method in the in-situ transparent method of the organ chip in the prior art.

In order to make the above and other objects, features and advantages of the present invention more comprehensible, several embodiments accompanied with figures are described in detail below to illustrate the light transparent method for organ-chip and its application.

Example 1

The sample of this example 1 was a tumor micro-organ self-cultured on a microplate (Nunclon Sphera wells, nunclon Sphera treated, U-bottom microplate accession number 174929) provided by thermo company, by the following method: inoculating 5 ten thousand tumor cell suspensions per milliliter into each well, 200 micro per well, and centrifuging at 500G, wherein the tumor cells are hct116 transfected with GFP protein, and the culture medium is rpmi1640; the sample is processed by an online transparent method, which comprises the following steps:

(1) Preparing corresponding transparent reagent: 1mL of the 60wt% medical iodixanol injection and DMEM culture solution are mixed at a ratio of 1:1, and then a pipetting gun is used for mixing uniformly to obtain a transparent reagent. The metal ion concentration in the finally obtained transparent reagent is: the concentration of potassium ions was 1.8mM; the concentration of calcium ions was 0.2mM; the concentration of magnesium ions was 0.2mM and the concentration of sodium ions was about 80mM.

(2) And (3) transparency: the tumor micro-organ was removed and immersed in an optically transparent reagent for 1 hour.

(3) And (3) observation: when the tumor micro-organ is placed under the imaging equipment for observation, the transparent reagent of the embodiment can be used for observing before and after the tumor micro-organ is transparent, the imaging signal intensity is improved as shown in fig. 6 (before the content (a) of fig. 6 is transparent and after the content (b) of fig. 6 is transparent), and the tumor cells can keep the living body state, so that all physiological functions are not affected.

(4) And (5) recovering: the tumor micro-organ is transferred to the culture medium environment and put back into the incubator, and can also maintain normal proliferation and growth.

Example 2

The embodiment utilizes a self-made chip culture device to carry out on-line transparency and observation on the cultured tumor organ chip by an on-line transparency method, and specifically comprises the following steps:

(1) And (3) manufacturing an adaptive chip: drawing photoetching drawings of chip intermediate layers corresponding to the first pattern layer 2 and the second pattern layer 3, manufacturing corresponding masks, forming corresponding male dies on a silicon wafer through soft photoetching, pouring PDMS prepolymer into the silicon wafer, curing for 20min in an oven at 80 ℃, taking down the PDMS blocks with patterns formed after curing, and cutting into proper sizes to obtain the first pattern layer 2 and the second pattern layer 3; sequentially bonding all layers of components according to the sequence of the glass slide, the connecting layer, the first pattern layer 2, the second pattern layer 3, the cover glass, the boss of the main channel liquid inlet hole, the boss of the middle liquid inlet hole and the boss of the middle liquid outlet hole, filling 0.01gPDMS prepolymer in the holes of the first pattern layer 2, centrifuging to form a U-shaped bottom surface, and curing again; after curing, the corresponding microfluidic devices are connected to obtain the microfluidic chip shown in fig. 2.

(2) Artificial micro-organ cultivation: 1 million/mL of tumor cell suspension, hct116 transfected with GFP protein, in RPMI1640 medium, was introduced into the chip, and centrifuged at 400G for 5 minutes, followed by stationary culture.

(3) Preparing corresponding transparent reagent: mixing 1mL of 60% iodixanol injection with standard DMEM culture solution at a ratio of 1:1, and then uniformly mixing by a pipette, wherein the concentration of metal ions in the finally obtained transparent reagent is as follows: the concentration of potassium ions was 1.8mM; the concentration of calcium ions was 0.2mM; the concentration of magnesium ions was 0.2mM and the concentration of sodium ions was 80mM.

(4) And (3) transparency: the optically transparent reagent obtained above was filled in a microfluidic pump, and after the bubbles in the chip were exhausted, the optically transparent reagent was continuously poured into the chip at a rate of 40. Mu.L/min for half an hour.

(5) And (3) observation: after the perfusion, the chip was moved to a microscope for observation, as shown in fig. 7, where content (a) is an imaging image of different depths before light transparency and content (b) is an imaging image of different depths after light transparency. It can be seen that the tumor micro-organ in the chip becomes transparent gradually after the light is transparent, and meanwhile, the structure of the tumor micro-organ can still be kept intact, and the imaging depth is obviously improved.

(6) And (5) recovering: the culture solution was continuously poured into the chip at a rate of 40. Mu.L/min for 60 minutes, and then returned to the incubator.

Example 3

The sample of this example is a heart micro-organ (produced and supplied by the medical instruments institute of su zhou, university of southeast) and is processed by an on-line transparent method, comprising the steps of:

(1) Preparing an ion concentration adjusting reagent: to the double distilled water, 3.5mM potassium chloride, 0.8mM calcium nitrate and 0.4mM magnesium chloride were added to a volume of 10mL.

(2) Preparing a light transparent reagent: mixing 1mL of 60% medical iodixanol injection (sigma) with RPMI1640 culture solution according to a volume ratio of 1:1.4, uniformly mixing by a pipette, adding 10 mu L of the ion adjusting reagent to adjust the ion concentration to obtain the final calcium ion concentration of 0.8mM and the potassium ion concentration of 3.5mM; the magnesium ion concentration was 0.4mM and the sodium ion concentration was about 90mM.

(3) And (3) transparency: the heart micro-organ was immersed in the light transparent agent for 30min.

(4) And (3) observation: when the heart micro-organ is placed under an imaging device for observation, as shown in fig. 8 (content (a) indicates before transparency and content (b) indicates after transparency), a difference in cell distribution inside the heart micro-organ which is not observed before transparency can be observed, and the heart micro-organ can be kept beating when transparent.

(5) And (5) recovering: the heart micro-organ is transferred to the culture environment and returned to the incubator.

Example 4

The sample of example 4 is a brain-like sample (produced and provided by the Wohwise biomedical corporation) and is processed by an on-line transparent method, which comprises the following steps:

(1) Preparing an ion concentration adjusting reagent: to double distilled water was added 1.8mM potassium chloride, 0.4mM calcium nitrate and 0.4mM magnesium chloride, 0.5. Mu.M zinc sulfate, and the volume was set to 10mL.

(2) Preparing a light transparent reagent: 1mL of 60% iodixanol injection (sigma) and F12 culture solution are mixed according to the volume ratio of 1:2, and the concentration is adjusted to 20%. Then, the mixture was homogenized by a pipette, and 10. Mu.L of the reagent for adjustment prepared in the step (1) was added to obtain a transparent reagent having a calcium ion concentration of 0.4mM, a potassium ion concentration of 1.8mM, a magnesium ion concentration of 0.4mM, a zinc ion concentration of 0.5. Mu.M and a sodium ion concentration of 100mM.

(3) And (3) transparency: the brains were immersed in the light transparent reagent for 8h.

(4) And (3) observation: the brains are placed under the imaging equipment for observation, details which cannot be observed before can be seen in the observation, and meanwhile, the brains can keep normal functions, and nerve discharge is not affected.

(5) And (5) recovering: the brains were transferred to the culture environment and returned to the incubator.

Comparative example 1

The sample is a heart micro-organ (produced and provided by the medical instruments institute of su zhou, university of southeast) and is processed by an on-line transparent method, which specifically comprises the following steps:

(1) Preparing a light transparent reagent: 60% of medical iodixanol injection (sigma) is taken and mixed with RPMI1640 culture solution according to a ratio of 1:1.4, and then a pipetting gun is used for mixing uniformly, so as to obtain the transparent reagent. No additional metal ions are introduced into the transparent reagent, and the ion concentration in the obtained reagent is as follows: the concentration of potassium ions was 1.6mM; the concentration of calcium ions was 0.5mM; the concentration of magnesium ions was 0.2mM, and the concentration of sodium ions was 90mM, and these metal ions were metal ions contained in the culture broth itself.

(2) And (3) transparency: the heart micro-organ was immersed in the light transparent agent for 30min.

(3) And (3) observation: the heart micro-organ is placed under an imaging device for viewing. The observed image is clearer.

(4) And (5) recovering: the heart micro-organ is transferred to the culture environment and returned to the incubator. Since the metal ions required for regulating the osmotic pressure of the organ-chip and/or maintaining the physiological functions of the organ-chip are not additionally introduced into the transparent reagent, the pulsation of the heart micro-organ is irreversibly damaged.

Comparative example 2

The sample is a heart micro-organ (produced and supplied by the medical instruments institute of su zhou, university of southeast), and the sample is processed by the following steps:

(2) Preparing a light transparent reagent: mixing 1mL of 60% medical iodixanol injection (sigma) with RPMI1640 culture solution according to a volume ratio of 1:1, uniformly mixing by a pipette, adding 10 mu L of the ion adjusting reagent to adjust the ion concentration to obtain the final calcium ion concentration of 0.8mM and the potassium ion concentration of 3.5mM; the magnesium ion concentration was 0.4mM and the sodium ion concentration was 80mM.

(4) And (3) observation: the heart micro-organ is placed under an imaging device for viewing. The other conditions were the same as in example 3 except that no personalized iodixanol concentration for the heart micro-organ was used due to the different volume ratio of iodixanol injection and culture solution, no clear imaging was observed, and the imaging effect was poor.

Comparative example 3

The sample is brain-like (produced and provided by wuhan Rui biological medicine company), and the sample is processed by the following steps:

(1) Preparing a light transparent reagent: 60% of medical iodixanol injection (sigma) is taken and mixed with DMEM culture solution in a ratio of 1:2, and then a pipetting gun is used for mixing uniformly to obtain the transparent reagent. The ion concentration of the obtained transparent reagent is as follows: the concentration of potassium ions was 1.2mM; the concentration of calcium ions was 0.26mM; the concentration of magnesium ions was 0.26mM and the concentration of sodium ions was 100mM. These metal ions are metal ions in the culture solution, and no metal ions are added additionally.

(2) And (3) transparency: the brains were immersed in the light transparent reagent for 8 hours.

(3) And (3) observation: the brain-like is placed under an imaging device for viewing. A clearer image can be observed.

(4) And (5) recovering: the brains were transferred to the culture environment and returned to the incubator. Failure to maintain proper concentration of metal ions while transparent may be manifested as inhibition of electroencephaloid emission, and inhibition of brain-like proliferation upon prolonged observation.

Comparative example 4

(1) Preparing an ion concentration adjusting reagent: to double distilled water was added 1.8mM potassium chloride, 0.2mM calcium nitrate and 0.2mM magnesium chloride, 5. Mu.M zinc sulfate, and the volume was set to 10mL.

(2) Preparing a light transparent reagent: 1mL of 60% medical iodixanol injection (sigma) and F12 culture solution are mixed according to a volume ratio of 1:1, then a pipette is used for mixing uniformly, and then 10 mu L of the adjusting reagent prepared in the step (1) is added to obtain a transparent reagent, wherein the concentration of calcium ions is 0.4mM, the concentration of potassium ions is 1.8mM, the concentration of magnesium ions is 0.4mM mu M, the concentration of zinc ions is 100mM.

(3) And (3) transparency: the brains were immersed in the light transparent reagent for 8 hours.

(4) And (3) observation: the brain-like is placed under an imaging device for viewing. The other conditions were the same as in example 4 except that the volume ratio of iodixanol injection and culture solution was different, and clear images could not be observed because the personalized iodixanol concentration for brains was not adopted, and the imaging effect was poor.

It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.

Claims

1. An on-line light transparent reagent of an organ-chip is characterized by comprising a culture solution for culturing the organ-chip, iodixanol and metal ions for regulating osmotic pressure of the organ-chip and maintaining physiological functions of the organ-chip; wherein the mass percentage of iodixanol in the transparent reagent is 10% -35%;

The total concentration range of the metal ions is 50-150 mM;

when the organ chip is a heart organ chip, the mass percentage of iodixanol in the transparent reagent is 24% -26.1%; the concentration of calcium ions in the transparent reagent is 0.75-0.85mM, and the concentration of potassium ions is 3.45-3.55mM; the concentration of magnesium ions is 0.35-0.45mM, and the concentration of sodium ions is 55-100mM;

When the organ chip is a brain-like organ chip, the iodixanol accounts for 17.1-24% of the transparent reagent; the transparent reagent has calcium ion concentration of 0.35-0.45mM, potassium ion concentration of 1.95-2.05mM, magnesium ion concentration of 0.35-0.45mM, zinc ion concentration of 4.5-5.5 μm, and sodium ion concentration of 50-100mM;

when the organ chip is a tumor chip, the mass percentage of iodixanol in the transparent reagent is 27.3% -33.3%, the concentration of calcium ions in the transparent reagent is 0.35-0.45mM, the concentration of potassium ions in the transparent reagent is 1.95-2.05mM, the concentration of magnesium ions is 0.35-0.45mM, and the concentration of sodium ions in the transparent reagent is 70-80mM.

2. The optically transparent reagent according to claim 1, wherein the culture medium is DMEM medium, F12 medium, MEM medium, RPMI1640 medium or a derivative medium of the above medium.

3. A method for on-line optical transparency of an organ-chip using the optically transparent reagent of claim 1 or 2, comprising the steps of:

(1) After bubble removal treatment is carried out on the organ chip, the optically transparent reagent as claimed in claim 1 or 2 is poured into the organ chip at a speed of 2-200 mu L/min for 0.4-0.6 hours; or immersing the artificial micro-tissue in the organ chip in the light transparent reagent for 0.8-1.2 hours; making the organ chip transparent to obtain a transparent organ chip;

4. A method as claimed in claim 3, further comprising the step of: